NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis

Analogs of diadenosine tetraphosphate (Ap4A).

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5'''-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase

7 pages, 5 figures, 2 tables.In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1, P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2–3 μM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12–20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates: diadenosine 5',5"'-P1, P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1, P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1, P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide {p4A, dATP, adenosine 5'-[α,β-methylene]-triphosphate, (Ap[CH2]pp), (S)-adenosine-5'-[α-thio]triphosphate ((Sp)ATP[αS]) and GTP}, luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1, P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1, P4-[α,β-methylene] tetraphosphate (Ap[CH2]ppA), (Sp-diadenosine 5',5"'-P1, P4-[α-thio]tetraphosphate ((Sp)Ap4A[αS]) and adenosine-5',5"'-P1, P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact α-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1, P3-triphosphate (Ap3A) or adenosine-5',5"'-P1, P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.This investigation was supported by grants from the Fundación Ramon Areces, Dirección General de Investigación Científica y Técnica (Grant PB87/0288) and from the Polish Academy of Sciences (within Project CPBR 3.13.4.4.4).Peer reviewe

Selective splitting of 3'-adenylated dinucleoside polyphosphates by specific enzymes degrading dinucleoside polyphosphates

8 pages, 3 figures, 1 table.Several 3'-[32P]adenylated dinucleoside polyphosphates (NpnN'p*As) were synthesized by the use of poly(A) polymerase (Sillero MAG et al., 2001, Eur J Biochem.; 268 : 3605-11) and three of them, ApppA[32P]A or ApppAp*A, AppppAp*A and GppppGp*A, were tested as potential substrates of different dinucleoside polyphosphate degrading enzymes. Human (asymmetrical) dinucleoside tetraphosphatase (EC 3.6.1.17) acted almost randomly on both AppppAp*A, yielding approximately equal amounts of pppA + pAp*A and pA + pppAp*A, and GppppGp*, yielding pppG + pGp*A and pG + pppGp*A. Narrow-leafed lupin (Lupinus angustifolius) tetraphosphatase acted preferentially on the dinucleotide unmodified end of both AppppAp*A (yielding 90% of pppA + pAp*A and 10 % of pA + pppAp*A) and GppppGp*A (yielding 89% pppG + pGp*A and 11% of pG + pppGp*A). (Symmetrical) dinucleoside tetraphosphatase (EC 3.6.1.41) from Escherichia coli hydrolyzed AppppAp*A and GppppGp*A producing equal amounts of ppA + ppAp*A and ppG + ppGp*A, respectively, and, to a lesser extent, ApppAp*A producing pA + ppAp*A. Two dinucleoside triphosphatases (EC 3.6.1.29) (the human Fhit protein and the enzyme from yellow lupin (Lupinus luteus)) and dinucleoside tetraphosphate phosphorylase (EC 2.7.7.53) from Saccharomyces cerevisiae did not degrade the three 3'-adenylated dinucleoside polyphosphates tested.This investigation was supported by grants from Dirección General de Investigación Científica y Técnica (PM198/0129 and BMC 2002-00866) and Comunidad de Madrid (08-1/0021-1/2001), and by grant PBZ-KBN 059/T09/04 from the State Committee for Scientific Research (KBN, Poland) to A.G.Peer reviewe